The proteins present in foods are precursors of a large amount of peptides with some type of special biological activity, with useful properties on different processes of the organism. Due to the enormous interest in developing natural products which have some type of “extra” beneficial effect for the health of the person who consumes them, obtaining some of these biopeptides formed from different plant raw materials, it would be a good chance to develop new functional ingredients or products. The possibility of generating these peptides is a field of state of the art research in the nutraceutical industry because it allows generating new applications in functional foods, giving added value of food components and byproducts, improving the nutritive properties of conventional foods and developing new dietary supplements or even new medicinal products.
Some byproducts of the agro-food industry have a high content of proteins and bioactive peptides which provide added value to the same. There are different mechanisms for favoring the increase of peptides by considerably enriching a matrix and assigning it determined characteristics, such as for example, antihypertensive, antihyperglycemic, antineurodegenerative, anticariogenic, and even antihyperlipidemic characteristics. With the use of proteolytic enzymes, proteins can be hydrolyzed at specific points, capable of generating a broad spectrum of peptides in the hydrolyzed products obtained with multiple physiological effects.
The present invention is focused on obtaining cocoa extracts rich in peptides with ACE and PEP enzyme inhibitory activity.
The angiotensin converting enzyme (ACE) catalyzes the conversion of inactive angiotensin I into angiotensin II, which is a strong vasoconstrictor, so one of the current therapies used in the treatment of hypertension consists of the administration of drugs inhibiting this enzyme. In recent years, due to the side effects caused by drugs, natural inhibitors which would help in controlling hypertension in a less aggressive manner have been described. Among others, Takayanagi et al. (“Angiotensin I converting enzyme-inhibitory peptides from wine”; Am. J. Enol. Vitic, 50:65-68 1999) describe ACE inhibitory peptides from wine and Wu et al. (“Hypotensive and physiological effect of angiotensin converting enzyme inhibitory peptides derived from soy protein on spontaneously hypertensive rats”; J. Agric Food Chem, 49:501-506 2001) describe soy peptides which also have an inhibitory effect on ACE. Many other works, such as that of Pedroche et al. (“Utilisation of chickpea protein isolates for production of peptides with angiotensin I-converting enzyme (ACE)-inhibitory activity”; Journal of the Science of Food and Agriculture, 82:960-965 2002); Tomita et al. (“Potent Antibacterial Peptides Generated by Pepsin Digestion of Bovine Lactoferrin”; J Dairy Sci, 74:4137-4142 1991); Fujita et al. (“LKPNM: a_product-type ACE-inhibitory peptide derived from fish protein”; Immunopharmacology, 82:960-965 2002); Pihlanto-Leppalla et al. (“Angiotensine I-converting enzyme inhibitory properties of whey protein digests: concentration and characterization of active peptides”; J Dairy Research, 67:53-64 2000), and Yamamoto (Yamamoto., “Antihypertensive Peptides Derived from Food Proteins”; Biopolymers, 43(2): 129-134 1997), assert that peptides derived from different food matrices produce the inhibition of the angiotensin converting enzyme. Thus, document JP 6128287 relates to obtaining a peptide which inhibits the activity of the angiotensin and is obtained by hydrolysis of a milk protein; and JP 188282 describes twenty-three tripeptides which are useful as antihypertensive agents and are obtained by treating sardine muscles with proteases.
Document ES 2 253 036 B1 describes bioactive peptides derived from egg white proteins by means of enzymatic hydrolysis. Said peptides have angiotensin converting enzyme inhibitory activity (ACE-inhibitory activity) in vitro and/or antihypertensive activity in rats and/or antioxidant activity. The whole hydrolyzed products, the low molecular weight fractions thereof, and their constituent peptides could be used as therapeutic substances with ACE-inhibitory and/or antihypertensive and/or antioxidant activity.
Among the patent documents which mention peptides obtained from cocoa, WO 02/42327 A2 describes obtaining and purifying 2S albumin from cocoa beans. The enzymatic hydrolysis of proteins, which generates aroma, peptide and amino acid precursors which give rise to a cocoa aroma after heating with sugar.
Though some authors find ACE inhibitors in chocolate, this inhibition is generally associated with polyphenols, as is the case of Actis-Goretta et al. (“Inhibition of Angiotensin Converting Enzyme Activity by Flavonol-Rich Foods”; Agric. Food Chem, 54:229-234 2006) and of patent document WO 01/41775 A2, which describes the use of cocoa polyphenols, specifically procyanidins, in the modulation of inflammatory pathways, in the maintenance of vascular health in mammals and as antibacterial treatments. However, patent document JP 2008019228 describes an ACE inhibitor for food use and for treating hypertension comprising a composition of amino acids derived from cocoa extracts from which the polyphenols have previously been extracted. Said document does not specify the peptide/amino acid sequence nor does it give its size range, nor does it mention any type of enzymatic hydrolysis in the process of isolating and/or purifying the extracts having ACE inhibitory activity.
Prolyl endopeptidase (PEP) activity is related to memory loss and learning processes since it degrades neuropeptides rich in proline, such as vasopressin and the substance P involved in these processes. Some studies also indicate that this enzyme could be related to Alzheimer's disease. Until now, different authors, such as Kim et al. (“Prolyl Endopeptidase Inhibitors from Green Tea”; Arch Pharm Res, 24 (4):292-296 2001) and Tezuka et al. (“Screening of crude drug extracts for prolyl endopeptidase inhibitory activity”; Phytomedicine, 6(3): 197-203 1999), assert that the polyphenols of certain plant extracts cause prolyl endopeptidase inhibition. Patent document US 2007/0116779 also describes the use of cocoa beans as a source of polyphenols, as one of the elements of pharmaceutical compositions, as an inhibitor of certain enzymes involved in neurodegenerative diseases, such as Alzheimer's or Parkinson's.
Other authors, such as Maruyama et al. (“Prolyl Endopeptidase Inhibitory Activity of Peptides in the Repeated Sequence of various Proline-Rich proteins”; Journal of Fermentation and Bioengineering, 74:145-148 1992) and Asano et al. (“Inhibition of prolyl endopeptidase by synthetic peptide fragments of human β-casein”; Agric. Biol. Chem, 55(3):825-828 1991) argue that some peptide fragments inhibit the action of PEP, but until now no one has referred to cocoa peptides as inhibitory metabolites of said enzyme.
Therefore, due to the increasing greater demand for new ingredients and compounds with this type of inhibitory activity, given the healthy characteristics of cocoa, the possibility of finding bioactive peptides from their extracts having ACE activity has been considered, and several have surprisingly been found which not only have ACE inhibitory activity in vitro, but said bioactive peptides also have PEP inhibitory activity in vitro. Furthermore, they also show antioxidant activity in vivo according to tests with C. elegans. 